OBJECTIVES: Oral submucous fibrosis (OSF) is a chronic, progressive, and potentially malignant disease of the oral cavity. A previous study by our team found that the aberrant expression of tRNA-derived small RNA (tsRNA) was involved in the development of OSF, with tiRNA-Val-CAC-002 showing the most significant difference. This study aimed to investigate the effect of tiRNA-Val-CAC-002 on fibroblast activation and its underlying mechanisms, elucidate the pathogenesis of OSF, and explore new effective targets for OSF prevention and treatment. METHODS: RT-PCR was used to detect tiRNA-Val-CAC-002 expression in OSF and arecoline-treated fibroblasts. Western blotting, MDC staining, and transmission electron microscopy validated the effects of arecoline and 002 on fibroblast autophagy. Western blotting was used to explore the signaling pathways related to tiRNA-Val-CAC-002 in OSF. RESULTS: Arecoline promotes fibroblast (FB) activation by upregulating tiRNA-Val-CAC-002. Arecoline stimulation and tiRNA-Val-CAC-002 overexpression activated fibroblasts by promoting autophagy. tiRNA-Val-CAC-002 regulates PI3K/AKT by mediating ITGB3 expression. CONCLUSIONS: Arecoline upregulates tiRNA-Val-CAC-002 expression in fibroblasts. Moreover, tiRNA-Val-CAC-002 may activate the autophagy of fibroblasts in OSF by ITGB3/PI3K/AKT pathway regulation, promoting the expression of collagen fibers.